Effect Of Salt Curing On Phosphorylation Of Muscle Proteins

The effects of salt curing on phosphorylation of muscle proteins were studied to provide a new theoretical mechanism by which salting alters meat quality. The muscles at 24 h postmortem from Crossed sheep(small Tailed Han sheep ×large Tailed Han sheep) were selected for the experiment. The changes of global phosphorylation level of muscle proteins during salt curing and the effects of different salt content on global phosphorylation level of muscle proteins were studied. The effects of salting temperature on muscle protein phosphorylation were analyzed. The differential phosphoproteins induced by salt were studied by two-dimensional electrophoresis. Model systems in vitro were established to study the effects of salt on the activity of protein kinase A and alkaline phosphatase, to speculate the way which salt alters protein phosphorylation. The results are as follows:(1) The changes of global phosphorylation level of myofibrillar and sarcoplasmic proteins during salt curing and the effects of different salt content on global phosphorylation level of myofibrillar and sarcoplasmic proteins were investigated by SDS-PAGE electrophoresis and fluorescent staining. The results indicated that the global phosphorylation level of myofibrillar proteins was affected by salting time and salt content. The global phosphorylation level of myofibrillar proteins in 2 % salt group decreased significantly during the salting development(P<0.05), and it was the lowest at salting 16 h, while the control group had no significant change. The global phosphorylation level of myofibrillar proteins in salt groups was significantly lower than the control group, and 3 % salt group was the lowest. The global phosphorylation level of sarcoplasmic proteins was not affected by salting time and salt content. But salt affected the phosphorylation level of some structural proteins and glycolytic enzymes.(2) Effects of salting temperature on phosphorylation level of myofibrillar and sarcoplasmic proteins were investigated by SDS-PAGE electrophoresis and fluorescent staining. The results indicated that the global phosphorylation level of myofibrillar and sarcoplasmic proteins was affected by salting temperature. The global phosphorylation level of myofibrillar proteins of controlled freezing point treatment(-1 oC) was significantly higher than room temperature treatment(25 oC) at salting 8 h(P<0.05), which had no significant difference at salting 16 h and 24 h. The results of sarcoplasmic proteins were opposite, the global phosphorylation level of sarcoplasmic proteins of controlled freezing point treatment was significantly lower than 4 oC treatment and room temperature treatment at salting 8 h(P<0.05), which had no significant difference at salting 16 h and 24 h. The effect of salting temperature on the phosphorylation level of individual protein bands was different. The phosphorylation level of actin and adolase increased with the increase of salting temperature, while myosin and enolase decreased.(3) The differential phosphoproteins induced by salt were investigated by two-dimensional electrophoresis. Fifteen differential phosphoproteins were identified at 0 % salt group and 3 % salt group. The main differential phosphoproteins of sarcoplasmic proteins included glycogen phosphorylase, triosephosphate isomerase, creatine kinase and myoglobin. All the proteins are involved in glycolysis, except for myoglobin. The main differential phosphoproteins of myofibrillar proteins included troponin T, tropomyosin, myosin light chain and actin, which are the main proteins of myofibrillar proteins..(4) Model systems in vitro which the myofibrillar proteins were substrates, were established by adding protein kinase A, alkaline phosphatase and salt. The phosphorylation level of myofibrillar proteins were analyzed to reveal the way which salt affected protein phosphorylation. The global phosphorylation level of myofibrillar proteins had no significant difference after adding salt alone. Protein kinase A increased protein phosphorylation level, while protein kinase A and salt inhibited protein phosphorylation action. Alkaline phosphatase decreased protein phosphorylation level significantly, while alkaline phosphatase and salt inhibited protein dephosphorylation action. The results indicated that salt affected protein phosphorylation by inhibiting the activity of protein kinase A and alkaline phosphatase.All the results indicated that protein phosphorylation was affected by salting time, salt content and salting temperature. Salt affected the activity of protein kinase A and alkaline phosphatase,which revealed the way salt regulated protein phosphorylation.